6.7 Wall synthesis and remodelling

Newly synthesised chitin and glucan polysaccharides are either linear or
shapeless molecules; they are cross-linked together and to other polymers to
form the rigid three-dimensional network typical of the mature cell wall. The
enzymes that accomplish this are called transglycosidases;
these extracellular enzymes are either tethered to the
plasma membrane with GPI anchors or located within the cell wall itself. The
first cell wall-modifying transglycosidase to be recognised is a GPI-anchored
enzyme encoded by the genes GEL and PHR in Aspergillus and Candida
and GAS in Saccharomyces (Mouyna et al., 2013). This enzyme
splits a β1,3-glucan molecule internally and transfers the newly generated
reducing end to the nonreducing end of a different molecule; this elongates the
glucan chain. Transglycosidases able to cross-link β1,6-glucan and chitin are
also known (Arroyo et al., 2016); indeed, six families of conserved GPI
proteins of this sort have so far been identified as being common across fungal
species.

The transglycosidases are generally considered to be responsible for
strengthening, or rigidifying, the fungal cell wall through the cross-links
they generate. Cell wall hydrolases (like endo-β1,3-glucanase, and
chitinase) and deacetylases, on the other hand, can plasticise the rigid
cell wall. Chitinase is involved in bud separation in Saccharomyces,
and endoglucanase is essential for cytokinesis in Schizosaccharomyces.
In filamentous fungi, some models of apical wall synthesis suggest a
delicate balance between hydrolysis of established polymers and synthesis of
new wall material at the hyphal apex (see
Section 5.16),
although there is no clear evidence that cell wall hydrolases are required
for hyphal tip extension. Deacetylases do appear to be widespread, and in
zygomycetes, ascomycetes, and basidiomycetes substantial deacetylation of
chitin (which produces chitosan) occurs. Chitosan is a more flexible
molecule than chitin, and it is resistant to chitinases. Also, deacetylation
of the chitin in the walls of plant pathogens can prevent the host plant’s
receptors from recognising the pathogen and so delay the onset of plant
defences.

During active hyphal growth wall construction activity is concentrated at the
hyphal tip. Autoradiographic studies indicate that all synthesis of chitin and
glucans takes place within 10 µm of the apex of the hypha of Neurospora
crassa. The tip is highly plastic as the wall is laid down, but as walls
mature they become more rigid. The rigidity is provided by the cross-linking of
polymers to which reference is made above, thickening of fibrils and the
deposition of materials in the interfibrillar matrix. The process is highly
polarised, and reliant on maintenance of a positive turgor pressure within the
cytoplasm (Bartnicki-Garcia et al., 2000). Wall rigidification in
vegetative hyphae is a remarkably rapid process, taking only a couple of
minutes. An estimate of the time elapsing between the deposition of plastic wall
material at the hyphal tip and its subsequent rigidification at the base of the
elongation zone (indicated by the point at which the hypha first attains a
constant diameter) can be obtained by dividing the length of the tapered
extension zone by the rate of hyphal elongation (Trinci, 1978).

Apical growth of the hypha requires long-distance transport between the
subapical part and the apex of the tip cell. We have discussed some of the
mechanisms that may be involved in Chapter 5. These
mechanisms include cytoskeleton-based motors delivering vesicles containing
enzymes and substrates over long distances to the hyphal tip (Bartnicki-Garcia,
2006; Riquelme et al., 2007; Steinberg, 2007, 2011). In addition,
hyphal growth is accompanied by the secretion of exoenzymes that participate in
both lysis and synthesis of fungal cell wall components which make the wall in
apical regions flexible. Extension of the hypha can then occur as both turgor
and cytoskeleton-based cytoplasmic expansion push the cytoplasm against the
flexible apical wall. A summarised diagrammatic compilation of these events is
shown in Fig. 4. This, together with the discussion in
Section 5.16 should provide a sufficiently
detailed view of fungal extension growth.

Fig. 4. Cartoon representation of an overall molecular model of hyphal
growth. The key feature of hyphal apical growth is rapid movement towards
the apex of all the materials needed to create new wall, new membranes and
new cytoplasmic components. Most of these materials are exported in vesicles
by the endoplasmic reticulum (ER) and Golgi organelles, the vesicles being
delivered to the apical vesicle cluster (called the Spitzenkörper) along
microtubules powered by motor proteins of the kinesin and dynein families.

The Spitzenkörper organises the final distribution of microvesicles
along actin microfilaments to the plasma membrane at the extending tip.
Vesicle fusion with the membrane is enabled by t-SNARE and v-SNARE proteins.

Sterol-rich ‘lipid rafts’ at the hyphal tip could provide domains for
apical proteins like signalling and binding complexes and might facilitate
endocytosis.

Endocytosis at the hyphal tip is dependent upon actin patches where
myosin-1 polymerises actin into filaments that take the endocytotic vesicles
away from the membrane. The extreme apex of hyphal tips undergoes extensive
exocytosis, which is mainly devoted to synthesis of wall polymers outside
the membrane and wall construction and maturation.

Endocytosis features in the flanking regions of the hyphal tip, and this
both recycles membrane components (originally delivered as exocytotic
vesicles) and imports nutrients; both of which are transported to the
endomembrane system for sorting and appropriate use.

This figure also shows that (potentially many) subterminal hyphal cells
contribute to the apical migration of resources; (streams of) vesicles,
(trains of rapidly moving) vacuoles and mitochondria are all transported
towards the apex and this transport extends through hyphal septa.

Also note that the position of nuclear division spindles is probably
specified by interaction between astral microtubules and membrane-bound
dynein-dynactin complexes, and septal positioning is associated with rings
of actin microfilaments.

Remember: this IS a cartoon, no attempt is made to portray
relative scale or relative timing (some structures, like division spindles)
are more transient than others (like the Spitzenkörper). Also, everything
happens, quickly; in the text we show that 38,000 vesicles have to
fuse with the apical membrane each minute (that’s over 600 every second) to
support extension of each hyphal tip of Neurospora crassa when it
is growing at its maximum rate. See text of Chapters 5 and 6 for complete
explanation, and refer to Steinberg (2007; 2011)
and Rittenouret al. (2009).

We do not wish to over-emphasise apical wall growth here, because it is not
the end of the story for the fungal wall. The picture that has emerged for the
cell walls of yeasts and filamentous fungi alike is that the wall is an
extremely dynamic construction with its components maintained in continual
balance as wall enzymes repair and remodel the original wall. Cell wall damage
in budding yeast, Saccharomyces cerevisiae, triggers a salvage
mechanism called the cell-wall-integrity pathway consisting of at least 18
cell-wall-maintenance genes controlled by a single transcription factor and a
specific signal transduction pathway. Sequences belonging to this integrity
pathway are conserved in several yeasts and filamentous fungi (Bowman & Free,
2006; Klis et al., 2006; Gow et al., 2017).

We discuss the dynamic nature of the fungal cell wall elsewhere in this text
in discussions of:

hyphal and spore differentiation (CLICK
HERE to view the page in Chapter 9),

All these processes require that wall synthesis is restarted within a mature
wall at a very closely-controlled place and at a specific time. Another relevant
circumstance to be aware of is that two hyphal branches that must be joined
together will synthesise a joint wall, and the resultant join can be stronger
than the original hyphal walls (CLICK HERE
to view the page in Chapter 12). All such remodelling depends on the coordinated
activity of several glycoproteins already present within the wall structure. The
‘wall-associated enzymes’ involved include chitinases, glucanases and peptidases
(Adams, 2004; Seidl, 2008; Gow et al., 2017); enzymes that hydrolyse
and break down cell wall components, as well as glycosyltransferases which are
involved in the synthesis and cross-linking of wall polymers.

In addition to these instances of remodelling, there are many
observations of secondary hyphal walls being synthesised as
internal thickenings mostly made up of thick fibrils. These
are probably glucans accumulated as intermediate to long-term nutritional
reserves. The first detailed observations of this were made by Jos
Wessels in the 1960s (reviewed by Bartnicki-Garcia, 1999) who
showed that during the final stages of maturation of the Schizophyllum
commune fruit body when its nutritional support is completely
endogenous, requiring no external sources of nitrogen or carbon, an
alkali-insoluble cell wall component, which was called R-glucan,
was the main fraction of the wall to be broken down. R-glucan contained both
β1,6 and β1,3 linkages, and was distinct from S-glucan
which was alkali-soluble and constituted the bulk of the cell wall material
left after mobilisation of the R-glucan.

Studies of the mobilisation process indicated that cell wall degradation
correlated with cap development and was controlled by changes in the level of a
specific R-glucanase enzyme. It seems that while glucose remains available in
the medium carbohydrate is temporarily stored in the form of R-glucan in the
walls of mycelial and fruit body hyphae. During this phase of net R-glucan
synthesis the R-glucanase is repressed by glucose in the medium, but when this
is exhausted the repression is lifted, R-glucanase is synthesised and by
breaking down the R-glucan it provides substrate(s) specifically required for
fruit body development.

The story is slightly different in Coprinopsis cinerea. In this
organism glycogen seems to serve a similar sort of function to the R-glucan of
Schizophyllum commune during fruit body development (glycogen is an α1,4
/α1,6 linked glucan (see Breakdown of polysaccharide: starch and glycogen
section of Chapter 10, CLICK HERE to
view the page); the reason may be that fruit bodies of C. cinerea
develop much more rapidly than those of S. commune and the glycogen
represents a much more efficient transient reserve that enables large quantities
of sugar to be rapidly translocated through the fruit body with no disturbance
to solute balance. Glycogen is involved in various aspects of vegetative
morphogenesis in C. cinerea (Waters et al., 1975b; Jirjis &
Moore, 1976), but so are wall glucans.

Mycelium of C. cinerea forms multicellular sclerotia, about 250 µm
diameter, as resistant survival structures which pass through a period of
dormancy before utilising their accumulated reserves to ‘germinate’ by producing
a fresh mycelium. Glycogen is synthesised and accumulated in young sclerotia,
but is not the long term storage product. For long term storage much of the
carbohydrate is converted into a form of secondary wall material, probably
glucan. Cells in the central bulk of the sclerotium may become extremely
thick-walled, the primary walls being thickened on their inner surfaces
by loosely-woven and very large fibrils, the development of which coincides with
the gradual disappearance of glycogen from the cells (Waters et al.,
1972, 1975a & b).

What we mean by ‘thick walled’ is illustrated in the
electronmicrographs of Figs 5 to 9. Secondary wall is external to the plasma
membrane but internal to primary wall. The illustrations in Figs 7 to 9 make it
clear that the secondary walls can come to make up a very considerable
proportion of the cell volume. This inevitably constricts the protoplasm
to a correspondingly smaller central lumen, but the indications are that these
cells remain alive; being effectively dormant until the sclerotium is presented
with amenable growth conditions (Erentalet al., 2008).

Figs 5 to 9. A selection of transmission electronmicrographs (TEMs) of
submerged mycelium and sclerotia of Coprinopsis cinerea showing the
two main types of thick walled hyphal cells; rind cells with dense,
pigmented secondary walls of heavily-melanised glucan on outer and side
walls which make up a plate-like layer of protective rind which resists
environmental extremes, and the secondary walls of medullary cells which are
uniformly thickened around the cell with large, branched fibres of glucan.
This image, Fig. 5, shows a TEM of typical young vegetative hyphae from the
submerged mycelium of Coprinopsis cinerea. Hyphae shown in
longitudinal (top) and transverse sections. Note the thin (primary) hyphal
walls that characterise this undifferentiated tissue. Key: n = nucleus, nc =
nucleolus, nm = nuclear membrane, mi = mitochondrion, v = vacuole, er =
endoplasmic reticulum, gly = glycogen granules. Electronmicrograph by Henry
Waters.

Fig. 6. Secondarily-thickened walls of rind cells on the outside of
sclerotia of C. cinerea. These secondary walls provide a protective
layer and are heavily melanised. Electronmicrograph by Henry Waters.

Fig. 7. Secondarily-thickened walls of the central (medulla) region of a
young sclerotium showing a cell with a secondarily-thickened wall in
transverse section alongside many normal cells. This secondary wall is not
melanised; it is constructed of thick fibres of glucan and is eventually
recycled to provide carbohydrate resources when the sclerotium germinates.
The granules (labelled gly) are accumulations of glycogen, which forms a
short-term carbohydrate resource. Accumulation of glycogen occurs before
formation of the glucan fibres and as the fibres are formed the glycogen
content declines. Note the dolipore septum (ds) at top left.
Electronmicrograph by Henry Waters.

Fig. 8. Medullary cells with secondarily-thickened wall shown in
longitudinal section, illustrating that the thickening (and consequent
constriction of the cell lumen) is fairly uniform over the length of the
hypha, and that the thickening can cross and involve the dolipore septa.
Electronmicrograph by Henry Waters.

Fig. 9. Magnified images of a longitudinal section of a
secondarily-thickened wall showing its fibrillar structure. The region of
secondarily-thickened (glucan) fibres highlighted in the left hand image is
shown at extreme magnification on the right. Electronmicrographs by Henry
Waters.

Dormant sclerotia may survive for several years, being
protected by a rind composed of tightly-packed hyphal tips which develop another
form of secondary thickening which is a heavily-melanised thickened wall that
forms an impervious surface layer (Fig. 6). Melanin is a dark coloured pigment
(almost black at high concentration); it is a high molecular weight polymer of
phenolic and/or indolic compounds. Phenols have hydroxyl group(s) bonded
directly to aromatic hydrocarbons; indoles are heterocyclic organic compounds
containing nitrogen, indole itself consists of a six-membered benzene ring fused
to a five-membered nitrogen-containing pyrrole and has the formula C8H7N.
These are negatively charged hydrophobic pigments that protect the hyphae and
spores when they are cross linked into the cell wall structure. Normally, they
are so completely interlinked, in fact, that it is possible in the laboratory to
digest away all the other wall components to leave ‘melanin ghosts’
of the entire original cell wall (Dadachova et al., 2008). Natural
melanins are biocompatible conductors. Nanocomposites using melanin
nanoparticles extracted from squid inks have potential for use in bioelectronic
devices such as biosensors and implantable devices (Eom et al. 2017).
Fungal melanin ghosts, with their greater structural integrity, could have more
interesting applications.

Melanin is extremely resistant to chemical and enzymic
attack and contributes to virulence in many pathogenic fungi (for example
Paracoccidioides brasiliensis, Sporothrix schenckii,
Histoplasma capsulatum, Blastomyces dermatitidis, and
Coccidioides posadasii) by reducing the susceptibility of melanised
fungi to host defences and drugs (Taborda et al., 2008). Melanin
increases cell wall rigidity, enabling hyphae of black fungi to penetrate
host tissues and pigmented conidia or yeast cells to remain turgid when
desiccated. Fungi with melanised walls are also resistant to electromagnetic
and ionising radiations; the pigment seems to provide both physical
shielding (from UV light) and quenching of cytotoxic free radicals (caused
by ionising radiations). Although the detailed structure of melanin is
unknown, two main types of melanin are found in the fungal cell wall, which
are named after precursors in their biosynthetic pathways.
DHN-melanin is synthesised from 1,8- dihydroxynaphthalene; and
DOPA-melanin, which is synthesised from
3,4-dihydroxyphenylalanine (L-DOPA). In various fungi, there is evidence
that melanin can be cross linked to mannans, mannosylated proteins,
galactoxylomannan, chitin or chitosan (Gow et al., 2017).

Other cell wall pigments, the carotenoids (Chapter 10,
CLICK HERE to view page), also protect against UV radiation. In general,
mutants of the entomopathogenic fungus Metarhizium anisopliae with
white conidia are more sensitive to UV radiation than mutants with purple
conidia, which were more sensitive than mutants with yellow conidia, which in
turn were more sensitive than the green wild strain (Braga et al.,
2006). Wall pigments have a function; they’re not just pretty colours.